title: Application of dried spot cards as a rapid sample treatment method for determining hydroxytyrosol metabolites in human urine samples. Comparison with microelution solid-phase extraction.
abstract: Two different rapid sample pretreatment strategies, dried spot cards, and microelution solid-phase extraction plates (μSPE), with ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) have been developed and validated for the determination of hydroxytyrosol and its metabolites in spiked human urine samples. Hydroxytyrosol, hydroxytyrosol-3'-O-glucuronide, hydroxytyrosol-4'-O-glucuronide, hydroxytyrosol-3-O-sulphate, and homovanillic alcohol-4'-O-glucuronide were used as the target compounds. Using the FTA DMPK-A dried urine spot card under optimum conditions, with 5 μL of preconcentrated urine volume and 100 μL of methanol/water (50/50, v/v) as the elution solvent, the extraction recovery (%R) of the compounds studied was higher than 80%, and the matrix effect (%ME) was less than 8%. The stability of these cards and punching at the centre or side of the card were also studied, obtaining an excellent stability after 7 days of storage and complete homogeneity across the surface of the dried drop. The different μSPE parameters that affect the efficiency were also studied, and under optimum conditions, the %R and the %ME were higher than 70% and lower than 17%, respectively. The linearity range in dried urine spot cards was 2.5-20 μM for all the metabolites, with the exception of hydroxytyrosol-3-O-sulphate and hydroxytyrosol, which were 0.3-70 μM and 2.5-50 μM respectively. With regards to μSPE, the linearity range was 0.5-5 μM for all the studied compounds, except for hydroxytyrosol-3-O-sulphate, which was 0.08-5 μM. The quantification limits (LOQs) were 0.3-2.5 μM and 0.08-0.5 μM in dried spot cards and in μSPE, respectively. The two developed methods were then applied and compared for determining hydroxytyrosol and its metabolites in human 24 h-urine samples after a sustained consumption (21 days) of a phenol-enriched virgin olive oil. The metabolites identified were hydroxytyrosol in its glucuronide and sulphate forms, homovanillic alcohol in its glucuronide and sulphate forms, homovanillic acid sulphate and hydroxytyrosol acetate sulphate.
authors.affiliation: Food Technology Department, XaRTA-TPV, Agrotecnio Center, Escuela Técnica Superior de Ingeniería Agraria, University of Lleida, Av/Alcalde Rovira Roure 191, 25198, Lleida, Spain.
authors.forename: Aida
authors.initials: A
authors.lastname: Serra
authors.forename: Laura
authors.initials: L
authors.lastname: Rubió
authors.forename: Alba
authors.initials: A
authors.lastname: Macià
authors.forename: Rosa-M
authors.initials: RM
authors.lastname: Valls
authors.forename: Úrsula
authors.initials: Ú
authors.lastname: Catalán
authors.forename: Rafael
authors.initials: R
authors.lastname: de la Torre
authors.forename: Maria-José
authors.initials: MJ
authors.lastname: Motilva
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods; D059788:Dried Blood Spot Testing / Q000379:methods*; D006801:Humans; D000069463:Olive Oil; D010626:Phenylethyl Alcohol / Q000031:analogs & derivatives* / Q000302:isolation & purification / Q000378:metabolism / Q000652:urine; D010938:Plant Oils / Q000378:metabolism; D052616:Solid Phase Extraction / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D000069463:Olive Oil; D010938:Plant Oils; C005975:3,4-dihydroxyphenylethanol; D010626:Phenylethyl Alcohol